Peptidases have a central role in the degradation of proteins by hydrolyzing peptide bonds. For a review of peptidases see, Walling, L. L. & Gu, Y.-Q., AMINOPEPTIDASES, Taylor, A., ed, R. G. Landes Co. (1996), herein incorporated by reference. Based on the reaction catalyzed, these enzymes can be classified as endopeptidases, carboxypeptidases or aminopeptidases. Aminopeptidases cleave single amino acid residues at the free N-termini of peptides or proteins.
Aminopeptidases are ubiquitous enzymes with a wide variety of activities detected in animal, plant and prokaryotic cells. In plants, aminopeptidases have been implicated in the mobilization of storage proteins, the salvage of C and N from dying cells, and in the activation and inactivation of regulatory molecules, including peptide hormones and peptides involved in cell-cell interactions. The expression of some aminopeptidases have been found to relate to a plant's response to mechanical wounding, pathogen infestation and other environmental stimuli.
Aminopeptidases traditionally have been distinguished from each other by the N-terminal amino acid cleaved by the enzyme. Leucine aminopeptidase (LAP) was first characterized from bovine lens by Hanson, H., et al. in Z. Physiol. Chem. 348(6):689 (1967). Plant LAP cDNA has since been generated from Arabidopsis, potato and tomato.
In addition to substrate specificity, plant aminopeptidases, including LAP, can be characterized by their optimal pH for enzymatic activity (Mikola, L. & Mikola, J., PLANT PROTEOLYTIC ENZYMES, VOL. II, Dalling, M. J., ed., CRC Press, Boca Raton, Fla. (1986). While LAPs with pH optima in the neutral and alkaline pH ranges have been known, only recently have acidic LAPs been discovered. Two distinct cDNA's encoding acidic leucine aminopeptidase (LAP-A) were generated from tomato (Gu, Y.-Q., et al., J. Biol. Chem. 271(42):25880 (1996), herein incorporated by reference). The deduced genes for these separate proteins were found to be tightly linked and therefore named LapA1 and LapA2. The entire gene sequences were found to share 93% homology but only two amino acid substitutions were within the coding region. The LAP-A enzyme was found to be large, greater than 327 kD, with monomers of 55 kD and a pI of 5.9. Neutral leucine aminopeptidases of 55 kD and LAP-like proteins of higher molecular weight were also found in tomato leaves after wounding but these proteins were also present in healthy tissue and therefore do not appear to be related to wounding or exposure to environmental stimuli. As further proof of the divergence between LAP-A and the other LAP proteins, genomic DNA blots of both types of Lap genes do not cross-hybridize under reduced stringency hybridization conditions.
A model for mechanical plant wounding has been developed (Hildmann, T., et al., The Plant Cell 4:1157 (1992), herein incorporated by reference. The authors found that exogenous addition of the phytohormones, abscisic acid (ABA) and methyl jasmonate (MeJa), induced strong accumulation of LapA mRNA in the leaves of potatoes as well as other transcripts of genes activated by mechanical wounding or pathogen infestation, including proteinase inhibitors I and II. They hypothesized that endogenous ABA and MeJa are involved in mediating the systemic response to wounding. In tomatoes, Pearce, G., et al. in Science 253:895 (1991) discovered systemin, a peptide signal which mediates the systemic response to wounding by increasing proteinase inhibitor transcripts in leaves. More recently, MeJa has been used successfully to differentially induce LapA mRNA in tomato leaves (Gu, Y.-Q., et al., J. Biol. Chem. 271(24):25880 (1996).
Promoters are non-transcribed regions of nucleic acid which bind RNA Polymerase II and direct transcription of mRNA. For a general discussion of promoters, see Lewin, B., GENES, 5TH ED., Oxford University Press (1994) chap. 29, of which the relevant discussion of promoters is incorporated by reference.
A simple eukaryotic promoter includes the dinucleotide CA motif surrounded by pyrimidines; Py.sub.2 CAPy.sub.5. This sequence lies at the start point of transcription (-3-+5) and is termed the initiation sequence. However, most promoters consist of other sequences that bind transcription factors which modulate the action of RNA Polymerase II. The more common sequences found in promoters are the TATA box, found at approximately position -25, and at about 100 base pairs upstream from the start point, consensus sequences CAAT, GGGCGG and/or ATTTGCAT. It is not necessary for promoter sequences to contain all of these sequences, but virtually all eukaryotic promoters contain at least one of the above sequences.
Typically, an expression cassette to be introduced into a transgenic plant contains the nucleic acid sequence to be transcribed and a promoter to direct the transcription. The promoter can either be homologous, i.e., occurring naturally to direct the expression of the desired transgene or heterologous, i.e., occurring naturally to direct the expression of a nucleic acid derived from a gene other than the desired transgene. Fusion genes with heterologous promoter sequences are desirable, e.g., for regulating expression of encoded proteins. In some instances, the promoter may constitutively bind transcription factors and RNA Polymerase II. In other instances, a heterologous promoter may be desirable because it has sequences that bind transcription factors the naturally occurring promoter lacks.
As described, tomato LapA promoters are induced by a variety of stressful conditions. Therefore, isolated and identified LapA promoters from tomato would be desirable, e.g., for directing expression of heterologous proteins, in transgenic plants under environmental stress. The present invention provides for these and other features.